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Oct4 Dependence of Chromatin Structure Within the Extended Nanog Locus in ES Cells Dana N

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Oct4 Dependence of Chromatin Structure Within the Extended Nanog Locus in ES Cells Dana N Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION Oct4 dependence of chromatin et al. 2003; Mitsui et al. 2003). Embryos depleted of Nanog fail to mature past5dofdevelopment (Mitsui et structure within the extended al. 2003). Nanog lies in a highly conserved genomic locus Nanog locus in ES cells with a distinctive configuration. The Nanog gene is flanked by several other genes that are expressed early in Dana N. Levasseur,1 Jianlong Wang,1 development and/or involved in critical fate decisions. Michael O. Dorschner,2 Colinearity of the genes encoding Aicda, Apobec1, 2 GDF3, Dppa3 (also known as Stella and PGC7), and John A. Stamatoyannopoulos, and Nanog is conserved throughout orthologous regions in 1,3,4 Stuart H. Orkin rodents and primates. The Aicda and Apobec1 genes en- code DNA editing enzymes that may serve as epigenetic 1Division of Hematology-Oncology, Children’s Hospital and modifiers by selectively influencing the genomewide Dana Farber Cancer Institute, Harvard Medical School, pool of methylatable targets during early development. Harvard Stem Cell Institute, Boston, Massachusetts 02115, Dppa3 is a maternal effect gene required for normal de- USA; 2Department of Genome Sciences, University of velopment. Recent work has shown that Dppa3 protects Washington, Seattle, Washington 98195, USA; 3Howard against inappropriate DNA demethylation in the mater- Hughes Medical Institute, Boston, Massachusetts 02115, USA nal genome and at certain imprinted loci (Nakamura et Embryonic stem (ES) cells offer insight into early devel- al. 2007). GDF3 and Nanog are essential for ES cell self- renewal and contribute to early remodeling of the devel- opmental fate decisions, and their controlled differentia- oping embryo. Interestingly, the region occupied by tion may yield vast regenerative potential. The molecu- these early developmental modifiers comprises only 160 lar determinants supporting ES cell self-renewal are in- kb, or 0.004% of the entire genome. The organization of completely understood. The homeodomain proteins the cluster implies complex interactions that may pro- Nanog and Oct4 are essential for mouse ES cell self-re- vide a window into regulation of pluripotency-related newal. Using a high-throughput approach, we discovered genes. DNaseI hypersensitive sites and potential regulatory el- Expression of the genes within the Nanog locus has ements along a 160-kb region of the genome that in- been reported in several cancers. Amplification of the cludes GDF3, Dppa3, and Nanog. We analyzed gene ex- Nanog locus on chromosome 12 in humans has been pression, chromatin occupancy, and higher-order chro- found in essentially all germ cell tumors analyzed and coordinated overexpression of GDF3, Dppa3, and Nanog matin structure throughout this gene locus and found is observed in human seminomas and embryonal carci- that expression of the reprogramming factor Oct4 is re- nomas, as revealed by expression profiling. Additionally, quired to maintain its integrity. Nanog has been characterized as a marker for metastatic Supplemental material is available at http://www.genesdev.org. germ cell tumors (Korkola et al. 2006; Santagata et al. 2007). Received August 17, 2007; revised version accepted December Oct4 function is required during the earliest stages of 18, 2007. development, and its depletion results in differentiation of the future embryo into extraembryonic trophecto- derm. In concert with Nanog, Sox2, KLF4, and Myc, Oct4 can reprogram differentiated somatic cells to a plu- Embryonic stem (ES) cells, which are derived from the ripotent state (Okita et al. 2007; Wernig et al. 2007). inner cell mass (ICM) of the developing blastocyst, are Nanog participates in an autoregulatory circuit together endowed with unlimited self-renewal capacity, while with Oct4, Sox2 (Boyer et al. 2005; Loh et al. 2006), and they retain the potential to differentiate into all germ likely other transcription factors (Ivanova et al. 2006; layers. There is considerable interest in revealing the ge- Matoba et al. 2006; Wang et al. 2006) to interact with netic programs responsible for ES cell self-renewal, with genes in cis or trans that preserve self-renewal and en- the ultimate goal of directing differentiation to create a able differentiation into all three germ layers. Here, we renewable tissue source for cell therapy. Currently, describe initial efforts to dissect regulatory interactions knowledge of the precise genetic determinants respon- and mechanisms operative in the Nanog locus. Using a sible for self-renewal is incomplete. high-throughput quantitative chromatin profiling (QCP) As components critical for ES cell self-renewal, several approach, we discovered DNaseI hypersensitive sites transcription factors, often referred to as core pluripo- (HS) and potential regulatory elements along a 160-kb tency factors, are subjects of intense study. One such region of chromosome 6 that includes GDF3, Dppa3, and factor, Nanog, was identified by virtue of its ability to Nanog. We analyzed gene expression, chromatin occu- promote self-renewal independent of the leukemia in- pancy, and higher-order chromatin structure throughout hibitory factor (LIF)/gp130 signaling pathway (Chambers this locus. We demonstrate that genes within the Nanog locus are coregulated. Importantly, we show that several pluripotency-related nuclear factors co-occupy chroma- [Keywords: Nanog; Oct4; chromatin; reprogramming; self renewal] tin throughout the locus, and that Oct4 expression is 4Corresponding author. required to maintain chromatin structure. Gene expres- E-MAIL [email protected]; FAX (617) 632-4367. sion and chromatin structure within this locus may Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/ serve as a useful “molecular sensor” in cellular repro- gad.1606308. gramming studies. GENES & DEVELOPMENT 22:000–000 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Levasseur et al. tional start site is a strong candidate for a cis-control element of the gene. Many intergenic regulatory elements lie far upstream of the gene or gene loci that they control. A survey of the large intergenic regions separating Nanog from its neigh- boring genes revealed strong HSs located −47 kb, −45 kb, and −38 kb from the Nanog transcriptional start site and 26 kb downstream from Dppa3 (Fig. 1, boxed region). Consistent with the cell-specific nature of the chromatin structure of the entire Nanog locus, DNase hypersensi- tivity was not observed within 200 kb upstream of or downstream from the Nanog start site in a murine eryth- roleukemia (MEL) cell line (data not shown). Transcription factors involved in ES cell self-renewal bind HS throughout the Nanog locus Transcription factors including Nanog, Oct4, Sox2, and others (such as ZFP281 and Nac1) collaborate to main- tain ES cell pluripotency (Boyer et al. 2005; Loh et al. 2006; Wang et al. 2006). To determine the potential con- tribution of these factors toward transcriptional regula- tion, we examined chromatin occupancy of Oct4, Nanog, ZFP281, and Nac1 at HSs within the Nanog lo- Figure 1. DNaseI HS determination by QCP. (A) Genomic struc- cus. These factors bind at shared positions at an unex- ture of the Nanog locus. The intergenic cluster region is shown pectedly high frequency throughout the locus (Fig. 2). boxed. (B) Nanog regulatory elements are indicated with four peaks Binding was most significant at the Nanog −5-kb HS, of HS located at the −5-kb region. A novel regulatory element is also with near 100-fold enrichments over background in seen at +9 kb. (C,D) QCP profiles for Dppa3 and GDF3 are shown. Note the large tandem HS peak in the GDF3 intron located −99 kb chromatin immunoprecipitation (ChIP) assays. Al- from the start of Nanog transcription. Arrows at the promoters in- though Nanog and Sox2 binding were observed previ- dicate direction of transcription. Genomic positions relative to the ously at both the −5-kb region and proximal promoter Nanog start site are shown above each HS. Data were mapped to the (Loh et al. 2006), our findings indicate that Oct4 also March 2005 (mm6) genome assembly and are visualized within the binds the upstream region. The Nanog proximal pro- University of California at Santa Cruz genome browser (Kent et al. moter and GDF3 intronic HS also exhibited high-level 2002). binding by Oct4, Nanog, ZFP281, and Nac1. Multispe- Results and Discussion cies DNA alignments indicate conservation of potential Oct4-, Nanog-, and ZFP281-binding sites at the −99-kb DNaseI hypersensitivity signature extends over the GDF3 and −5-kb Nanog regulatory elements (Supple- entire region containing Nanog and the other early-expressed genes Little is known regarding the cis-acting elements respon- sible for Nanog regulation. A comprehensive survey of regulatory DNA within the Nanog locus has not been undertaken. DNaseI hypersensitivity is a sensitive indi- cator of regulatory elements in complex loci. As such, we mapped DNaseI hypersensitivity by QCP throughout the Nanog locus in mouse ES cells (Fig. 1; Dorschner et al. 2004). This high-throughput method permits assess- ment of HSs throughout the locus in an efficient manner and with great sensitivity. A strong HS was observed at −5 kb from the transcriptional start
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